The transition metal manganese plays a variety of important roles in biology and medicine. For example, a large number of enzymes use Mn(ll) in their catalytic centers. Along with Mn(ll), the higher oxidation states, Mn(lll) and Mn(IV), are also used in crucial oxygen-centered redox chemistry. Of particular interest is the unique water oxidation chemistry enabled by the tetranuclear Mn cluster of the oxygen evolving complex (OEC) of photosystem II. This cluster couples the high oxidation potential of a proximal tyrosine radical (Yz() to the oxidation of two bound waters, releasing molecular oxygen at the final step of a 5-intermediate cycle. Thus this system is an important example of metalloradical chemistry, and the fact that each state can be generated in high yield with laser flashes makes this photosynthetic system ideal for exploring this intriguing chemistry. We will examine the intermediates of the oxygen evolving cycle with multifrequency (9, 31, 35, and 130 GHz) advanced electron paramagnetic resonance (EPR) methods, including ENDOR, ESEEM, and HYSCORE. These experiments will target the structure of the Mn cluster, its amino acid coordination, the location and function of the Ca2+ and Cl- cofactors, and the binding of substrate waters. We will follow leads from new x-ray crystal structures to target specific issues of high current interest. We will use our high field/frequency (130 GHz) EPR/ENDOR instrument to perform high resolution spectroscopy of the Mn cluster in single crystals of photosystem II. Using a newly assembled rapid freeze quench system, we will cryotrap samples on the millisecond timescale after laser flash sequences, with the ultimate goal of characterizing the final short-lived S4-state of the OEC cycle. This research promises to reveal important new details concerning how a biological metal cluster can produce molecular oxygen from water with an efficiency far greater than we can achieve with our current technologies. Relevance: This grant proposal focuses on understanding this vital life process, which produces the oxygen of our atmosphere that we require for respiration, and biologically activates the electrons and protons from water needed by plants to convert atmospheric carbon dioxide into our primary food sources.